Enzymes are highly specific proteins and greatly reduce the activation energy needed in chemical reactions. In addition to its proteinaceous nature, the essence of an enzyme is its catalytic activity. The catalytic activity is characterized by the enzyme's substrates and products, the relationship among which, in turn, define the nature of the reaction catalyzed by the enzyme.
The structures of proteins are determined by genetic information, that is. information that each organism inherits in the form of molecules of deoxyribonucleic acid (DNA). DNA is made up of four types of nucleotides, each of which contains a nitrogenous base, a pentose sugar, deoxyribose, and a phosphate group. The nucleotides differ only in their nitrogenous bases. Oligonucleotides are linear sequences of nucleotides which are joined by phosphodiester bonds. The linear order of nucleotides in a DNA molecule determines the order of amino acids in a protein. The amino acid composition of a protein might be referred to as its zero-order structure and this composition is partially responsible for a protein's net charge and solubility. The amino acids are strung together via amide or peptide bonds and the sequence of amino acids and location of disulfide bridges, if there are any, are termed its primary structure. The primary structure is thus a complete description of the covalent connections of the protein and is at least indirectly responsible for the higher levels of structure and for all properties of the protein, including enzymatic activity. Secondary structure refers to the steric relationship of amino acid residues that are close to one another in the linear sequence. Tertiary structure refers to the steric relationship of amino acid residues that are far apart in the linear sequence. Proteins that contain more than one polypeptide chain display an additional level of structural organization, namely quaternary structure, which refers to the way in which the chains are packed together. A protein's genetic code is characterized by the relationship between the sequence of bases in its DNA and the sequence of its amino acids. Chromosomes, which are threadlike structures consisting of DNA and proteins, carry genes, the units of heredity.
Mutations are inheritable changes in the genetic material, they occur when the position of a nucleotide or of a segment of several nucleotides in the DNA chain is altered, when nucleotides or segments of DNA are added or removed, or when one nucleotide is changed to another. Mutations may result from mistakes in DNA replication or they may be caused by any one of a number of mutagenic agents, which may act by breaking the DNA molecule or by changing the molecular structure of a nucleotide. Since the structure of DNA is responsible for the sequence of amino acids in proteins, mutations may cause changes in the protein for which a segment of DNA codes.
Mutations are not necessarily deleterious and, in fact, there are many reasons for wanting to mutate a gene. Classically, mutagenesis has been used to understand basic processes of genes and gene expression. The identification of genes in pathways, determinations with regard to control of genes, as well as gene mapping have all benefited from mutagenic methods. Recently, recombinant protein expression has made use of newer molecular techniques for mutagenesis. Mutant genes may be generated randomly using chemicals, UV irradiation, polymerase chain reaction (PCR) or by using specifically altered bacterial strains known as mutators. Mutations may also be made at predesignated places by site directed mutagenesis and with PCR.
With gene expression in mind, the goal of mutating a gene is generally to obtain an enzyme which has a desired characteristic the native enzyme lacks. It would be most advantageous to be able to synthesize enzymes or to change individual amino acid residues at will in order to obtain enzymes having specific features imparted to them which, in turn, would give those enzymes greater commercial value. Some examples of such desirable traits would include, but would not be limited to, creation of enzymes having (a) heat stability; (b) increased activity at elevated temperatures; (c) stability or increased activity in a narrow or wide pH range; (d) increased or decreased expression levels; (e) improved solubility; (f) increased binding affinities; (g) recognition of different or modified sequence or receptor sites and (h) exhibition of heterobifunctional activity (ability to work two different ways possibly at the same time).
Endonucleases are enzymes that catalyze breaks in DNA strands at relatively specific sites in specific nucleotide groups. Such restriction endonucleases, or restriction enzymes, provide a means for reproducibly cutting up a molecule into recognizable pieces. A good example of the important role played by restriction enzymes, and the potential commercial impact of their successful synthesis, is illustrated in Strand Displacement Amplification (SDA) developed by G. Terrance Walker, Melinda S. Fraiser, James L. Schram, Michael C. Little, James G. Nadeau and Douglas P. Malinowski, NucleicAcid Research 1992, Vol. 20, No. 7, 1691-1696. SDA is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo.sup.- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as targets for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank 1992 Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5' and 3'-ends that can then undergo SDA. Newer target generation schemes eliminate the requirement for restriction enzyme cleavage of the target sample prior to amplification. The methods exploit the strand displacement activity of exo.sup.- klenow to generate target DNA copies with defined 5' and 3'-ends. The target generation processes occur at a single temperature, after initial heat denaturation of the double-stranded DNA. Target copies generated by these processes are then amplified directly by SDA. The target generation processes can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5' and 3'-sequences modified as desired. It would be most advantageous to be able to perform SDA at higher temperatures as such performance would result in increased specificity, decreased background and improved amplification.
Enzyme activity has been shown to be influenced by many factors, including temperature, pH, substrate and buffer. And, while proteins are known to be intrinsically unstable against heat, the temperature range of thermal stability and the rate of thermal inactivation are very different for different enzymes. Similarly, optimum pH range for a given enzyme's activity varies greatly between different enzymes and may also be dependent upon temperature. Choice and concentration of substrate are of significant importance when trying to determine rate of catalysis since different substrates are converted at different rates. Additionally, rate of catalysis may be impacted by the type of buffer selected.
Enzymatic activity has also been known to be influenced by inhibitors and activators, which are commonly known as "effectors". For example, activator-type effectors may have to be added to an assay mixture to achieve maximum activity whereas inhibitor-type effectors may be present either in the sample or in the reagents or may arise during the reaction in the case of product inhibition. And, while most enzyme-catalyzed reactions show an initial velocity proportional to concentration of enzyme, there are some instances where the relationship does not hold true, as may be the case where small amounts of an enzyme inhibitor is present.
Immunochemical assays generally fall into one of two classifications. In the competitive assay, a limited quantity of binding material is contacted with a solution containing the analyte and a known concentration of a labeled analytc. The labeled and unlabeled analyte compounds compete for the binding sites on the binding material. By reference to a calibration curve, the amount of labeled analyte bound to the binding material can be correlated with the concentration of the analyte in the test solution. A second type of immunological assay, the sandwich assay, involves contacting a binding material with a solution containing the analyte to cause the analyte to bind to the binding material. This complex is then contacted with a solution of a labeled binding material, generally an antibody, which reacts with the bound analyte. The amount of bound labeled binding material is directly proportional to the amount of bound analyte. The sandwich-type assay is generally limited to antigens large enough to accommodate binding of two antibodies simultaneously, one of them being labeled with the marker enzyme. Enzyme-labeled antibodies may be used for the non-radioactive detection of many other classes of biomolecules, such as specific DNA sequences, using DNA-oligonucleotide hybrid formation as the specific recognition principle or carbohydrate residues of glycoproptein, using carbohydrate-lectin binding.
Enzymes to be used in substrate assays have to fulfill a number of quality criteria concerning: (a) specificity, which is the absence of side activities towards other substrates which may be present in the sample or assay mixture; (b) purity, which is the absence of contaminating activities or other contaminants interfering with the analytical and detection systems, not necessarily purity with respect to the absence of other inactive proteins; (c) stability of the test mixture during the assay reaction as well as on long-term storage: (d) kinetic properties: (e) pH optimum; (f) solubility and surface properties which refers to the absence of interference by adsorption or aggregation effects; and (g) cost.
In enzyme immunoassays, enzymes are used as markers for the detection of antibody or hapten-antigen interactions and thus compete with other labeling principles, such as radioisotopes or physicochemical labels. Measurements based on immune reactions have become increasingly popular for determination of high (e.g. catalytically inactive proteins) as well as low molecular weight substances. Conjugates of the marker enzyme with an antibody or hapten may be synthesized by chemical coupling with several bifunctional reagents according to the nature of the reactive groups on the enzyme. Compared to radioimmunoassays, the use of enzymes as labels has the advantage that contact with radioactive materials is avoided, the label may be easily detected with commonly available laboratory equipment and the reagents have a much longer shelf-life.
In each assay method, the unbound labeled material has to be separated from the bound labeled material. A widely used technique for such separation is to immobilize one of the reactants. For instance, an antibody may be adsorbed onto a solid support such as a test tube wall. After labeled material and analyte become bound to the immobilized antibody, the solid support is rinsed free of unbound labeled material.
Most enzyme engineering has been carried out by site-directed mutagenesis following extensive and detailed structural characterization of the enzyme. In nearly all cases this requires a high-resolution three-dimensional structure determined by X-ray crystallography. Mutagenesis of enzymes of unknown chemical structure has been accomplished, by necessity, via random processes. Rellos and Robert K. Scopes, Protein Fxpression and Purification 1994, Vol. 5, 270-277, describe the creation of random mutagens of Zymomonas mobilis alcohol dehydrogenase-2 using polymerase chain reaction techniques. Genetic engineering methods have allowed the production of large quantities of known enzymes and have also enabled the utilization of site-directed mutagenesis.
These processes require selection procedures that must screen thousands of mutants for the desired property. What the art lacks is a relatively simple selection procedure to enable the successful screening of large numbers of mutant enzymes. The method of the present invention recognizes the commercial value associated with enzymatic engineering and provides a novel method to successfully screen for desired mutants. According to the present invention, enzymatic mutagenesis is effected randomly, that is via PCR, chemical or mutator strain. From such treatments, a library of mutations is generated from which the desired mutation must be sorted from the wild type and other types of unwanted mutants. Thus, the gene must be phenotypically expressed so that it may be identified. Without such phenotypic expression, mutations may only be determined through sequencing. The particular phenotype sought will be expressed by the ability of a specific enzyme to cleave an oligonucleotide under non-native conditions, that is, those conditions not normally encountered in nature.